Expression and Purification of G Protein-Coupled Receptor (GPCR) Transmembrane Peptides

Abstract

Despite the role of G-protein-coupled receptors (GPCRs), the largest family of integral membrane proteins, in various physiological processes and significant interest by the pharmaceutical industry, our knowledge of this class of proteins is limited by the lack of structural and functional data. Recently, “divide and conquer” approaches to membrane protein structure determination, in which GPCRs are segmented into shorter and easier-to-handle transmembrane (TM) peptides, have yielded structure-function insights. Current methods for generating TM peptides, however, require mutations that ultimately may have a structural effect. Herein, we have generated a robust system for the high-level expression and purification of GPCR peptide fragments in E. coli that eliminates this requirement.The engineered system utilizes an N-terminal ketosteroid isomerase (KSI) domain, redundant Strep affinity tags, thrombin cleavage site, and a nickel affinity tag to enable the expression and rapid purification of receptor peptides for biophysical and structural characterization. The KSI domain directs fusion protein expression to inclusion bodies, where the fusion protein can be solubilized in detergent and purified by nickel affinity chromatography. The TM peptide is released from the fusion protein by thrombin digestion and separated by differential Strep affinity.Buffer conditions that are suitable for nickel affinity chromatography, thrombin cleavage, and strep affinity chromatography have been optimized, where purification yields for fusion proteins of human adenosine A2a receptor (hA2aR) TM 7, a model peptide, were ∼20 mg/L. Furthermore, hA2aR TM 7 peptide has been recovered following thrombin cleavage and subsequent purification in sufficient quantities for biophysical characterization. However, when this system was applied to the purification of fusion proteins of hA2aR TM 6-7, the observed purification yields were drastically reduced (∼1 mg/L). The role of hA2aR TM 6 and the generalizability of this approach to several GPCRs will be discussed.